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Background Mycobacterium abscessus (Mabs), a rapidly growing Mycobacterium species, is considered an MDR organism. Among the standard antimicrobial multi-drug regimens against Mabs, amikacin is considered as one of the most effective. Parenteral amikacin, as a consequence of its inability to penetrate inside the cells, is only active against extracellular mycobacteria. The use of inhaled liposomal amikacin may yield improved intracellular efficacy by targeting Mabs inside the cells, while reducing its systemic toxicity. Objectives To evaluate the colocalization of an amikacin liposomal inhalation suspension (ALIS) with intracellular Mabs, and then to measure its intracellular anti-Mabs activity. Methods We evaluated the colocalization of ALIS with Mabs in eukaryotic cells such as macrophages (THP-1 and J774.2) or pulmonary epithelial cells (BCi-NS1.1 and MucilAir), using a fluorescent ALIS and GFP-expressing Mabs, to test whether ALIS reaches intracellular Mabs. We then evaluated the intracellular anti-Mabs activity of ALIS inside macrophages using cfu and/or luminescence. Results Using confocal microscopy, we demonstrated fluorescent ALIS and GFP-Mabs colocalization in macrophages and epithelial cells. We also showed that ALIS was active against intracellular Mabs at a concentration of 32 to 64 mg/L, at 3 and 5 days post-infection. Finally, ALIS intracellular activity was confirmed when tested against 53 clinical Mabs isolates, showing intracellular growth reduction for nearly 80% of the isolates. Conclusions Our experiments demonstrate the intracellular localization and intracellular contact between Mabs and ALIS, and antibacterial activity against intracellular Mabs, showing promise for its future use for Mabs pulmonary infections.

ESX type VII secretion systems are complex secretion machineries spanning across the mycobacterial membrane and play an important role in pathogenicity, nutrient uptake and conjugation. We previously reported the role of ESX-4 in modulating Mycobacterium abscessus intracellular survival. The loss of EccB4 was associated with limited secretion of two effector proteins belonging to the WXG-100 family, EsxU and EsxT, and encoded by the esx-4 locus. This prompted us to investigate the function of M. abscessus EsxU and EsxT in vitro and in vivo. Herein, we show that EsxU and EsxT are substrates of ESX-4 and form a stable 1:1 heterodimer that permeabilizes artificial membranes. While expression of esxU and esxT was up-regulated in M. abscessus-infected macrophages, their absence in an esxUT deletion mutant prevented phagosomal membrane disruption while maintaining M. abscessus in an unacidified phagosome. Unexpectedly, the esxUT deletion was associated with a hyper-virulent phenotype, characterised by increased bacterial loads and mortality in mouse and zebrafish infection models. Collectively, these results demonstrate that the presence of EsxU and EsxT dampens survival and persistence of M. abscessus during infection.

Mycobacterium abscessus is the most pathogenic species among the predominantly saprophytic fast-growing mycobacteria. This opportunistic human pathogen causes severe infections that are difficult to eradicate. Its ability to survive within the host was described mainly with the rough (R) form of M. abscessus, which is lethal in several animal models. This R form is not present at the very beginning of the disease but appears during the progression and the exacerbation of the mycobacterial infection, by transition from a smooth (S) form. However, we do not know how the S form of M. abscessus colonizes and infects the host to then multiply and cause the disease. In this work, we were able to show the hypersensitivity of fruit flies, Drosophila melanogaster, to intrathoracic infections by the S and R forms of M. abscessus. This allowed us to unravel how the S form resists the innate immune response developed by the fly, both the antimicrobial peptides- and cellular-dependent immune responses. We demonstrate that intracellular M. abscessus was not killed within the infected phagocytic cells, by resisting lysis and caspase-dependent apoptotic cell death of Drosophila infected phagocytes. In mice, in a similar manner, intra-macrophage M. abscessus was not killed when M. abscessus-infected macrophages were lysed by autologous natural killer cells. These results demonstrate the propensity of the S form of M. abscessus to resist the host’s innate responses to colonize and multiply within the host.

Non-tuberculous mycobacteria (NTM) are a large group of micro-organisms comprising more than 200 individual species. Most NTM are saprophytic organisms and are found mainly in terrestrial and aquatic environments. In recent years, NTM have been increasingly associated with infections in both immunocompetent and immunocompromised individuals, prompting significant efforts to understand the diverse pathogenic and signalling traits of these emerging pathogens. Since the discovery of Type VII secretion systems (T7SS), there have been significant developments regarding the role of these complex systems in mycobacteria. These specialised systems, also known as Early Antigenic Secretion (ESX) systems, are employed to secrete proteins across the inner membrane. They also play an essential role in virulence, nutrient uptake and conjugation. Our understanding of T7SS in mycobacteria has significantly benefited over the last few years, from the resolution of ESX-3 structure in Mycobacterium smegmatis , to ESX-5 structures in Mycobacterium xenopi and Mycobacterium tuberculosis . In addition, ESX-4, considered until recently as a non-functional system in both pathogenic and non-pathogenic mycobacteria, has been proposed to play an important role in the virulence of Mycobacterium abscessus ; an increasingly recognized opportunistic NTM causing severe lung diseases. These major findings have led to important new insights into the functional mechanisms of these biological systems, their implication in virulence, nutrient acquisitions and cell wall shaping, and will be discussed in this review.

Free-living amoebae are thought to represent an environmental niche in which amoeba-resistant bacteria may evolve towards pathogenicity. To get more insights into factors playing a role for adaptation to intracellular life, we characterized the transcriptomic activities of the emerging pathogen Mycobacterium abscessus in amoeba and murine macrophages (Mϕ) and compared them with the intra-amoebal transcriptome of the closely related, but less pathogenic Mycobacterium chelonae. Data on up-regulated genes in amoeba point to proteins that allow M. abscessus to resist environmental stress and induce defense mechanisms, as well as showing a switch from carbohydrate carbon sources to fatty acid metabolism. For eleven of the most upregulated genes in amoeba and/or Mϕ, we generated individual gene knock-out M. abscessus mutant strains, from which ten were found to be attenuated in amoeba and/or Mϕ in subsequence virulence analyses. Moreover, transfer of two of these genes into the genome of M. chelonae increased the intra-Mϕ survival of the recombinant strain. One knock-out mutant that had the gene encoding Eis N-acetyl transferase protein (MAB_4532c) deleted, was particularly strongly attenuated in Mϕ. Taken together, M. abscessus intra-amoeba and intra-Mϕ transcriptomes revealed the capacity of M. abscessus to adapt to an intracellular lifestyle, with amoeba largely contributing to the enhancement of M. abscessus intra-Mϕ survival., Author summary The emerging pathogen Mycobacterium abscessus differs from other rapidly growing mycobacteria by its ability to survive phagocytosis by human macrophages. These virulence traits render M. abscessus pathogenic in vulnerable hosts with structural lung disease. How patients become infected with M. abscessus remains unclear, as M. abscessus is not or only very rarely isolated from the environment. Co-culturing of M. abscessus and amoeba increases the virulence of the bacteria for subsequent mouse infection, but little is known about M. abscessus virulence in general. Until now, the prediction of M. abscessus virulence factors was essentially based on the comparative analysis of the genome of M. abscessus with that of Mycobacterium chelonae, a closely related mycobacterium that occasionally causes opportunistic human infections, but to a much lesser extent than M. abscessus. Here, by transcriptomic and functional approaches, we decipher a global view on M. abscessus adaptation to intracellular life.

Free-living amoebae might represent an evolutionary niche. In order to get more insights into the potential amoebal training ground forMycobacterium abscessus, we characterized its full transcriptome in amoeba (Ac) and macrophages (Mφ), as well as theMycobacterium chelonaeintra-Ac transcriptome for comparison. Up-regulated genes in Ac allowedM. abscessusto resist environmental stress and induce defense mechanisms, as well as showing switch from carbohydrate carbon sources to fatty acid metabolism. Eleven genes implicated in the adaptation to intracellular stress, were mutated, with all but one confirmed to be involved inM. abscessusintra-Mφ survival. Cloning two of these genes inM. chelonaeincreased its intra-Mφ survival. One mutant was particularly attenuated in Mφ that corresponded to the deletion of an Eis N-acetyl transferase protein (MAB_4532c). Taken together,M. abscessustranscriptomes revealed the intracellular lifestyle of the mycobacteria, with Ac largely contributing to the enhancement ofM. abscessusintra-Mφ survival.

Mycobacterium abscessus is a peculiar rapid-growing Mycobacterium (RGM) capable of surviving within eukaryotic cells thanks to an arsenal of virulence genes also found in slow-growing mycobacteria (SGM), such as Mycobacterium tuberculosis. A screen based on the intracellular survival in amoebae and macrophages (MΦ) of an M. abscessus transposon mutant library revealed the important role of MAB_0855, a yet uncharacterized Mycobacterial membrane protein Large (MmpL). Large-scale comparisons with SGM and RGM genomes uncovered MmpL12 proteins as putative orthologs of MAB_0855 and a locus-scale synteny between the MAB_0855 and Mycobacterium chelonae mmpL8 loci. A KO mutant of the MAB_0855 gene, designated herein as mmpL8(MAB), had impaired adhesion to MΦ and displayed a decreased intracellular viability. Despite retaining the ability to block phagosomal acidification, like the WT strain, the mmpL8(MAB) mutant was delayed in damaging the phagosomal membrane and in making contact with the cytosol. Virulence attenuation of the mutant was confirmed in vivo by impaired zebrafish killing and a diminished propensity to induce granuloma formation. The previously shown role of MmpL in lipid transport prompted us to investigate the potential lipid substrates of MmpL8(MAB). Systematic lipid analysis revealed that MmpL8(MAB) was required for the proper expression of a glycolipid entity, a glycosyl diacylated nonadecyl diol (GDND) alcohol comprising different combinations of oleic and stearic acids. This study shows the importance of MmpL8(MAB) in modifying interactions between the bacteria and phagocytic cells and in the production of a previously unknown glycolipid family.

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

International audience; Mycobacterium abscessus, a rapidly growing mycobacterium (RGM) and an opportunistic human pathogen, is responsible for a wide spectrum of clinical manifestations ranging from pulmonary to skin and soft tissue infections. This intracellular organism can resist the bactericidal defense mechanisms of amoebae and macrophages, an ability that has not been observed in other RGM. M. abscessus can up-regulate several virulence factors during transient infection of amoebae, thereby becoming more virulent in subsequent respiratory infections in mice. Here, we sought to identify the M. abscessus genes required for replication within amoebae. To this end, we constructed and screened a transposon (Tn) insertion library of an M. abscessus subspecies massiliense clinical isolate for attenuated clones. This approach identified five genes within the ESX-4 locus, which in M. abscessus encodes an ESX-4 type VII secretion system that exceptionally also includes the ESX conserved EccE component. To confirm the screening results and to get further insight into the contribution of ESX-4 to M. abscessus growth and survival in amoebae and macrophages, we generated a deletion mutant of eccB4 that encodes a core structural element of ESX-4. This mutant was less efficient at blocking phagosomal acidification than its parental strain. Importantly, and in contrast to the wild-type strain, it also failed to damage phagosomes and showed reduced signs of phagosome-to-cytosol contact, as demonstrated by a combination of cellular and immunological assays. This study attributes an unexpected and genuine biological role to the underexplored mycobacterial ESX-4 system and its substrates.

The coevolution of mycobacteria and amoebae seems to have contributed to shaping the virulence of nontuberculous mycobacteria in macrophages. We identified a pool of genes essential for the intracellular survival of Mycobacterium abscessus inside amoebae and macrophages and discovered a hot spot of transposon insertions within the orthologous ESX-4 T7SS locus. We generated a mutant with the deletion of a structural key ESX component, EccB4. We demonstrate rupture of the phagosomal membrane only in the presence of an intact eccB4 gene. These results suggest an unanticipated role of ESX-4 T7SS in governing the intracellular behavior of a mycobacterium. Because M. abscessus lacks ESX-1, it is tempting to speculate that ESX-4 operates as a surrogate for ESX-1 in M. tuberculosis.

Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium involved in pulmonary and cutaneo-mucous infections worldwide, to which cystic fibrosis patients are exquisitely susceptible. The analysis of the genome sequence of M. abscessus showed that this bacterium is endowed with the metabolic pathways typically found in environmental microorganisms that come into contact with soil, plants, and aquatic environments, where free-living amoebae are frequently present. M. abscessus also contains several genes that are characteristically found only in pathogenic bacteria. One of them is MAB_0555 , encoding a putative phospholipase C (PLC) that is absent from most other rapidly growing mycobacteria, including Mycobacterium chelonae and Mycobacterium smegmatis . Here, we report that purified recombinant M. abscessus PLC is highly cytotoxic to mouse macrophages, presumably due to hydrolysis of membrane phospholipids. We further showed by constructing and using an M. abscessus PLC knockout mutant that loss of PLC activity is deleterious to M. abscessus intracellular survival in amoebae. The importance of PLC is further supported by the fact that M. abscessus PLC was found to be expressed only in amoebae. Aerosol challenge of mice with M. abscessus strains that were precultured in amoebae enhanced M. abscessus lung infectivity relative to M. abscessus grown in broth culture. Our study underlines the importance of PLC for the virulence of M. abscessus . Despite the difficulties of isolating M. abscessus from environmental sources, our findings suggest that M. abscessus has evolved in close contact with environmental protozoa, which supports the argument that amoebae may contribute to the virulence of opportunistic mycobacteria.

Mycobacterium abscessusis a pathogenic, rapidly growing mycobacterium responsible for pulmonary and cutaneous infections in immunocompetent patients and in patients with Mendelian disorders, such as cystic fibrosis (CF).Mycobacterium abscessusis known to transition from a smooth (S) morphotype with cell surface-associated glycopeptidolipids (GPL) to a rough (R) morphotype lacking GPL. Herein, we show thatM. abscessusS and R variants are able to grow inside macrophages and are present in morphologically distinct phagosomes. The S forms are found mostly as single bacteria within phagosomes characterized by a tightly apposed phagosomal membrane and the presence of an electron translucent zone (ETZ) surrounding the bacilli. By contrast, infection with the R form leads to phagosomes often containing more than two bacilli, surrounded by a loose phagosomal membrane and lacking the ETZ. In contrast to the R variant, the S variant is capable of restricting intraphagosomal acidification and induces less apoptosis and autophagy. Importantly, the membrane of phagosomes enclosing the S forms showed signs of alteration, such as breaks or partial degradation. Although not frequently encountered, these events suggest that the S form is capable of provoking phagosome–cytosol communication. In conclusion,M. abscessusS exhibits traits inside macrophages that are reminiscent of slow-growing mycobacterial species.

Mycobacterium abscessus is a rapidly growing mycobacterium, causing opportunistic infections in humans, and notably pulmonary infections. M. abscessus is able to multiply inside macrophages (MФ) and environmental amoebae. Here we demonstrate that M. abscessus undergoes adaptations in amoebae allowing its survival in MФ. Intracellular adaptations of M. abscessus to amoebae and MФ were assessed by RNAseq. We observed a significant enrichment of biological pathways reflecting adaptations to oxidative stress and metabolic adaptations illustrated by the consumption of fatty acids and activation of the sulfate assimilation pathway. These adaptations have been described in intramacrophagic Mycobacterium tuberculosis, a strictly pathogenic mycobacteria infecting the lung of humans and causing tuberculosis. Among the set of genes induced by M. abscessus during the amoebal co-culture are genes implicated in polyamine transport, MoCo (molybdopterin cofactor) biosynthesis and iron-sulfur (Fe-S) cluster assembly. The induction of such genes, described as virulence factors from intracellular bacteria, might enhance M. abscessus virulence and thus allow its survival in MФ. Forty-five genes are highly induced along the amoebal co-culture. In comparison, the amoebal co-culture with Mycobacterium chelonae, a mycobacterium that belongs to the same genomic complex as M. abscessus and causing solely extrapulmonary infections, does not elicit the same adaptations; potentially explaining M. chelonae inability to persist in macrophages. Five operons, representing a total of 10 genes, were deleted from M. abscessus genome by homologous recombineering. These genes are required for both M. abscessus survival in amoebae and MФ. Overexpression of two of these genes in M. chelonae, MAB_1517c and MAB_2649, encoding a TcmP (tetracenomycin polyketide synthesis O-methyltransferase) protein and an MmpS (mycobacterial membrane protein small) protein respectively, enhances M. chelonae survival in MФ, suggesting that the induction of these genes favors M. abscessus survival in MФ. Analyses of M. abscessus transcriptome in MФ also shed light on adaptations specific to the bacterium intramacrophagic life. Several genes highly induced in macrophages are implicated in biological pathways known to contribute to bacteria virulence, including proline metabolism, protein secretion by the type II secretion system and the MEP (methylerythritol phosphate) pathway. Among the set of induced genes selected according to their level of induction and their biological activity, N-acetylation and redox activities, bounding to oxygen and detoxification from nitric oxide by dioxygenases are significantly enriched. Among operons from this set of genes, it appears that M. abscessus eis2 gene (MAB_4542c), encoding a N-acetyltransferase, is essential for M. abscessus survival in MФ.In addition, a complementary approach to RNAseq, the screening of a transposon (Tn) mutant library of M. abscessus inside amoebae, revealed important roles of the mmpL8 gene encoding a mycobacterial membrane protein large belonging to a family of proteins implicated in lipid biosynthesis and export to the cell surface. When this protein was no longer produced by M. abscessus, a lower amount of a new glycolipd family (GDND, glycosyl diacetylated nonadecyl diol) was observed as well as a deleterious phenotype in MФ.To conclude, our work has shown a fundamental role of amoebae in triggering the virulence of M. abscessus, further allowing its survival in macrophages. Besides, three genes that have been studied more extensively – mmpL8, eis2 and eccB4 (revealed by the Tn library screening) – are required for M. abscessus survival in macrophages and confirmed its pathogenic behavior., La mycobactérie à croissance rapide Mycobacterium abscessus est un pathogène opportuniste de l’homme, particulièrement des poumons, capable de se multiplier au sein de macrophages (MФ) mais aussi d’amibes environnementales. Nous avons pu montrer que l’environnement amibien est propice à l’adaptation de M. abscessus à cette survie intramacrophagique. Par des analyses transcriptomiques comparant les adaptations de M. abscessus au sein d’amibes ou de MФ, nous observons des enrichissements de voies biologiques démontrant des adaptations au stress oxydatif et des adaptations métaboliques telle que la béta-oxydation des lipides et l’assimilation des sulfates. Ces adaptations ont également été observées chez Mycobacterium tuberculosis, mycobactérie pathogène pulmonaire stricte de l’homme responsable de la tuberculose. Parmi les gènes induits par M. abscessus au sein des amibes figurent des gènes impliqués dans le transport de polyamines, la biosynthèse du MoCo (molybdopterin cofactor) ainsi que l’assemblage des centres fer-soufre (Fe-S). L’induction de tels gènes, décrits comme des facteurs de virulence chez certaines bactéries intracellulaires, contribuerait à la virulence de M. abscessus en permettant sa survie intramacrophagique. Quarante-cinq gènes ont été identifiés comme fortement induits en amibes chez M. abscessus. Mycobacterium chelonae, appartenant au même complexe génomique que M. abscessus et responsable exclusivement d’infections cutanées, ne présente pas de telles inductions après analyse de son transcriptome intracellulaire, ce qui pourrait expliquer son absence de survie en MФ. Cinq opérons recouvrant 10 gènes ont été délétés au sein du génome de M. abscessus par recombinaison homologue. Ces gènes sont requis pour la survie de M. abscessus en amibes et en MФ. La surexpression chez M. chelonae de deux de ces gènes, MAB_1517c et MAB_2649, codant respectivement une protéine TcmP (tetracenomycin polyketide synthesis O-methyltransferase) et une MmpS (mycobacterial membrane protein small), a conféré une survie intra-macrophagique à M. chelonae, suggérant que l’induction de ces gènes en amibes favorise la survie intracellulaire de M. abscessus en MФ. Enfin, l’analyse du transcriptome de M. abscessus en MФ révèle des adaptations propres à la vie intramacrophagique. Différents gènes particulièrement induits sont impliqués dans le métabolisme de la proline, la sécrétion de protéine par le système de sécrétion de type II ou appartiennent la voie MEP (méthylérythritol phosphate), des voies biologiques contribuant à la virulence de pathogènes. De plus, parmi les gènes induits, certains correspondent à des activités de N-acétylation, d’oxydoréduction, de liaison à l’oxygène ou de détoxification de l’oxyde nitrique par des dioxygénases qui sont enrichies. Comparé à 5 autres opérons délétés, sélectionnés selon leur niveau d’induction et leur activité biologique, le gène eis2 (MAB_4532c), codant une N-acétyltransférase, est essentiel à la survie de M. abscessus en MФ.L’utilisation d’une approche complémentaire, le criblage d’une banque de mutants par transposition chez M. abscessus en amibes, a révélé le rôle essentiel du gène mmpl8 (mycobacterial membrane protein large 8) parmi la famille de protéines MmpL, impliquées dans le transport et/ou la synthèse de lipides mycobactériens. Chez M. abscessus, l’absence de production de cette protéine est corrélée à un défaut d’export d’un nouveau glycolipide (GDND, glycosyl diacetylated nonadecyl diol) et à un phénotype délétère pour la bactérie en MФ.En conclusion, notre travail a montré le rôle fondamental de l’amibe dans l’apprêtement de M. abscessus à la survie intramacrophagique. Trois gènes ayant fait l’objet d’études approfondies, mmpL8, eis2 ainsi que le gène eccB4 (MAB_3759c) mis en évidence par le crible amibien de la banque de mutants de transposition, participent à ce phénotype, confirmant le caractère pathogène de M. abscessus.

Mycobacterium abscessus is a fast growing mycobacterium, and an opportunistic pathogen responsible for lung infections particularly in patients with cystic fibrosis, and for mucocutaneous infections. The source of contamination could be environmental but human-to-human contaminations are not excluded. Environmental amoeba could play a role as a reservoir. M. abscessus is able to resist to the bactericidal defense mechanisms of environmental and human phagocytes. The complete genome of M. abscessus has been sequenced and presents numerous non-mycobacterial virulence factors. Some are known virulence factors in the bacterial world, such as phospholipase C or the magnesium uptake factor MgtC. These factors have been shown to be induced in the presence of amoeba, but cannot alone explain the intracellular survival and virulence of M. abscessus. We thus, in the course of this project, screened a library of mutants generated by transposition in M. abscessus, in search of mutants lacking intracellular growth in amoeba and macrophages. This approach made it possible to identify, in a major way, 5 genes of the ESX-4 locus of M. abscessus encoding a type VII secretion system with all these conserved core components. In order to better understand the contribution of ESX-4 to the intracellular survival of M. abscessus, a mutant obtained by double recombination within the eccB4 gene in the M. abscessus type strain (ΔeccB4) was constructed. EccB4 is a central structural element of the secretion system encoded by ESX-4. ΔeccB4 has a defect of survival within the cells, linked to deficiency of blockage of the phagosomal acidification as well as a defect of degradation of the phagosomal membrane, preventing phagosome-cytosol contact. This work made it possible to reveal for the first time in the mycobacterial world the role of an ancestral locus ESX-4 secretion within a mycobacterium. The study of the proteins secreted by this locus is currently underway in the laboratory, in order to consider therapeutic and vaccine approaches to counter this multiresistant antibiotic mycobacterium.; Mycobacterium abscessus est une mycobactérie à croissance rapide, et un pathogène opportuniste responsable d’infections pulmonaires notamment chez les patients atteints de la mucoviscidose, et d’infections cutanéomuqueuses. La source de contamination pourrait être environnementale mais des contaminations interhumaines ne sont pas exclues. Les amibes environnementales pourraient jouer un rôle de réservoir. M. abscessus est capable de résister aux mécanismes de défense bactéricides des phagocytes environnementaux et humains. Le génome complet de M. abscessus a été séquencé mettant en évidence de nombreux facteurs de virulence non mycobactériens. Certains sont des facteurs de virulence connus dans le monde bactérien, comme la phospholipase C ou le facteur de captation du magnésium MgtC. Ces facteurs ont été montré induits en présence d’amibes, mais ne peuvent à eux seuls expliquer la survie intracellulaire et la virulence de M. abscessus. Nous avons donc, au cours de ce projet, criblé une banque de mutants générée par transposition chez M. abscessus, à la recherche de mutants dénués de croissance intracellulaire en amibes et macrophages. Cette approche a permis d’identifier, de façon majeure, 5 gènes du locus ESX-4 de M. abscessus codant un système de sécrétion de type VII avec tous ces composants cœur conservés. Pour mieux comprendre la contribution d’ESX-4 dans la survie intracellulaire de M. abscessus, un mutant obtenu par double recombinaison au sein du gène eccB4 dans la souche type de M. abscessus (ΔeccB4) a été construit. EccB4 est un élément structurel central du système de sécrétion codé par ESX-4. ΔeccB4 présente un défaut de survie au sein des cellules, lié à déficit de blocage de l’acidification phagosomale ainsi qu’un défaut de dégradation de la membrane phagosomale, empêchant un contact phagosome-cytosol. Ce travail a permis de révéler pour la première fois dans le monde mycobactérien le rôle d’un locus ancestral de sécrétion ESX-4 au sein d’une mycobactérie. L’étude des protéines secrétées par ce locus est actuellement en cours au laboratoire, afin d’envisager des approches thérapeutiques et vaccinales pour contrer cette mycobactérie multirésistante aux antibiotiques.